Double salts of S-adenosil-L-methionine

ABSTRACT

New double salts extremely stable of S-Adenosil-L-methionine with sulphuric acid and p.toluensulphonic acid of the formula SAM + .HSO 4   - .H 2  SO 4 .2CH 3  C 6  H 4  SO 3  H and SAM + .HSO 4   - .H 2  SO 4 .CH 3  C 6  H 4  SO 3  H, process for the preparation thereof and therapeutic compositions containing them.

This invention relates to new enzymatic salts, a process for theirpreparation and the therapeutic compositions which contain them.

More precisely this invention relates to new extremely stable salts ofS-Adenosil-L-Methionine (SAM), to a process which enables it to beprepared simply and economically on an industrial scale and to newpharmaceutical compositions which contain them as the active principle,for use in numerous fields of human therapy.

SAM is notably a product of natural origin, found in all livingorganisms from bacteria to plants, from single cell organisms tosuperior mammals including man, the structure of which has been knownfor some time and is identified by the following formula: ##EQU1## inwhich X is a generic anion.

In living organisms SAM is formed by the intervention of enzymes(S-adenosilmethioninsynthetasis or S-adenosiltransferasis) in thecytoplasmatic ambit starting from methionine assumed with the nutrimentsor from ATP present as energy reserve in every living cell.

It has also been known for some time that SAM is a product offundamental importance in a large number of biological reactions ofenzymatic transmethylation, because of which it has always beenconsidered a very important reagent in biochemistry. The big problemwith this substance has however always been its extreme instability atambient or above ambient temperatures, and its methods of productionwhich are laborious and cannot easily be carried out on an industrialscale.

In recent years, research directed towards stabilising SAM to such anextent as to make it possible to use it in the field of biologicalresearch has been directed towards the preparation of salts which arestable under normal temperature and humidity conditions.

In this way the chloride and sulphate of SAM have been produced, butthey are of use only as reagents in biochemistry for short times,because even in the dry state their stability is limited in time at lowtemperatures. Furthermore their preparation processes are useful for theproduction of small quantities, but certainly not for production on anindustrial scale.

We have now completely unexpectedly found new salts of SAM which areindefinitely stable with time at temperatures up to 45°C, and which canbe prepared by a new process easily carried out economically on anindustrial scale, giving high yields. These salts have provedsurprisingly to possess strong curative power in many fields of humantherapy, often apparently without correlation between them.

The new salts according to the present invention are double salts of SAMwith p-toluenesulphonic acid and sulphuric acid, corresponding to theformula SAM⁺.HSO₄ ⁻.H₂ SO₄.2CH₃ C₆ H₄ SO₃ H, and SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃C₆ H₄ SO₃ H respectively.

The great technical progress achieved by these new salts can be seenfrom the following table which compares the stability with time, at45°C, in the dry state, of the two most stable salts of SAM known up tothe present time, i.e. the chloride and sulphate, with the newdi-sulphate, di-p.toluenesulphonate. The figures refer to the percentageof SAM residue after the times indicated:

                  TABLE 1                                                         ______________________________________                                        Anion         30 d     60 d     120 d  180 d                                  Chloride     20       --       --     --                                      Sulphate     50       5        --     --                                      Di-sulphate di-p.                                                                          100.1    99.9     100.2  100.4                                   toluensulphonate                                                              ______________________________________                                    

The stability values obtained with the di-sulphatemono-p.toluenesulphonate are completely equivalent to those reported forthe di-sulphate di-p.toluenesulphonate.

The process for preparing the new salts according to the inventioncomprises essentially the following stages:

a. preparation of a solution rich in SAM either by extraction fromnatural substances which contain it or by enzymatic synthesis fromadenosin triphosphate (ATP) and methionine

b. precipitation of the SAM present in the filtered aqueous solution bya saturated aqueous solution of picrolonic acid or by solutions of thesame acid in organic solvents soluble in water such as methyl, ethyl,propyl, isopropyl, n-butyl, or iso-butyl alcohols; or acetone,methylethylketone, methylisobutylketone, ethyl acetate, tetrahydrofuran,2-methoxyethanol, 2-ethoxyethanol, dioxan or dimethylformamide.

c. dissolving the filtered precipitate in a mixture consisting of equalparts by volume of a solvent partially miscible with water such asmethylethylketone, methylisobutylketone, n-butanol or isobutanol and asolution of equal normality of p-toluensulphonic acid and sulphuricacid.

d. separation of the organic layer and addition to the aqueous solutionof an organic ketone or alcohol solvent completely soluble in water.

e. redissolving the precipitate in a 10-20% solution ofn-toluenesulphonic acid in methanol and treating the solution withdecolouring charcoal.

f. addition to the concentrate of an organic solvent able to precipitatethe pure SAM in a well crystalline and easily filterable form.

As stated, stage (a) of the process can be carried out in different wayswhich are equally efficient for the purposes of obtaining a concentratedsolution of SAM.

According to one alternative yeast (Saccharomyces Cerevisiae, Torulopsisutilis, Candida utilis etc.), enriched in SAM by the addition ofmethionine under suitable conditions (Schlenk, Enzymologia 29, 283(1965)), with ethyl acetate and then with sulphuric acid having anormality between 0.1 and 0.5, preferably 0.35 N. at ambienttemperature, so as to cause the lysis of the cells and the passage intosolution of practically 100% of the SAM present.

Preferably quantities of water and acetate between 1/20 and 1/5 of theweight of the humid cells are used, and the treatment is protracted fora time between 15 and 45 minutes, preferably for 30 minutes.

Sulphuric acid is then added, and lysis is carried out for a timebetween 1 hour and 2 hours, preferably 1 hour and a half.

It should be noted that the lysis of the yeast cells conducted with amixture of organic solvent and dilute sulphuric acid is much moreconvenient than that normally carried out with perchloric acid atambient temperature, or with formic acid or acetic acid at 60°C and thelike, in that not only does it take place at ambient temperature, whichis very favourable to the stability of the SAM, but is conducted undersuch conditions that the solution can be easily filtered from thecellular residues, and does not contain any of the impurities which arepresent when the other lysant means are used, and which are difficult toeliminate with the known processes for preparing pure SAM.

According to a further alternative, the stage (a) of preparing the SAMfor enzymatic synthesis is carried out by the action of the enzymeATP-methionine-adenosiltransferasis (E.C. 2.4.2.12) on an incubationmixture containing adenosiltriphosphate (ATP) and methionine.

The essential condition for the purposes of the industrial execution ofthis method is that the enzyme is pure and is in a form which is easilyisolated both from the initial incubation mixture and from the SAMproduced.

The applicant has discovered a process for purifying the enzymeATP-methionine-adenosiltransferasis by chromatography by affinity, plusa column reaction method, which enable the aforesaid objects to beattained.

The affinity chromatography of the specific enzyme according to thepresent invention is carried out by percolating a solution containingit, for example a raw extract of yeast or "Escherichia coli", through acolumn filled with a support solid to which a group has been covalentlybonded which acts as a competitive inhibiter of the enzyme itself.

It has been surprisingly found that an excellent filling for such apurification column consists of an activated gel of polysaccharides towhich L-lysine has been covalently bonded. The affinity of the specificenzyme for the lysine residue bonded to the solid matrix causes a delayin elution of the enzyme by the column, and it is thus possible toobtain separation from the other proteins in a very pure form.

However the separation of the enzyme from the eluate which contains it,for use in the next enzymatic synthesis stage, has given completelyunsatisfactory results because once separated, its stability diminishedwith time, and in addition after being used only once in the synthesisof the SAM, it was destroyed in the subsequent SAM isolation operations.

The applicant has found that excellent results are obtained instead byabsorbing the eluate containing the specific enzyme in a suitablesupport solid and carrying out the catalytic reaction between methionineand ATP in the column, leading to the formation of SAM.

A suitable support solid consists of a polysaccharide activated by areagent suitable for bonding proteins to solid supports, such ascyanogen bromide.

By percolating a solution of ATP and methionine in a suitable buffersolution through the column, an eluate is obtained at the base of thecolumn containing the SAM.

The stage (b) of the process enables the SAM to be separated in a stateof high purity. In fact, in an acid environment the only compoundprecipitated by picrolonic acid in SAM, as is shown by thin layerchromatography in accordance with Anal. Biochem. 4, 16-28 (1971).

Picrolonic acid has thus an extremely and surprisingly selective action.

The other precipitating agents added up to the present, such as picricacid, Reinecke salt, or boric acid, give very impure precipitates whichalways require a subsequent purification of the SAM by ion exchangecolumn chromatography, a process which is extremely costly and difficultto carry out industrially. It is also difficult to obtain the productwith the necessary purity. The use of aqueous solutions of picrolonicacid or solutions of the same acid in the aforementioned organicsolvents does not present particular problems, and is an operation whichis carried out at ambient temperature.

The stage (c) is preferably carried out with solutions containingp-toluenesulphonic acid and sulphuric acid in concentrations bothbetween 0.05 and 0.2 N, preferably 0.1 N, and with an organic solventparticularly miscible with water such as methylethylketone or n-butanol.The use of the organic solvent enables the aqueous acid solutions to bevery much reduced and practically eliminates all the picrolonic acid.

The stage (d) of the process is carried out by preferably using between4 and 8 volumes (with respect to the volume of the aqueous solution) ofa solvent chosen from the group comprising acetone, methyl alcohol,ethyl alcohol and propyl alcohol.

It has also been surprisingly found that if in stage (e) the minimumquantity of methanol necessary to dissolve the precipitate originatingfrom stage (d) is used, the double salt SAM⁺.HSO₄ ⁻.H₂ SO₄.2 CH₃ C₆H.sub. 4 SO₃ H separates in the subsequent precipitation stage (f).

If however a volume of methanol equal to at least double the necessaryvolume is used in stage (e), the double salt SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃ C₆H₄ SO₃ H separates in the subsequent precipitation stage (f).

The use of intermediate quantities of methanol leads to the formation ofmixtures of the two salts.

The final precipitation of one or other of the new salts according tothe invention (stage f) requires the use of an organic solvent chosenfrom the group consisting of methanol, ether, chloroform, n-propanol,isopropanol, n-butanol, isobutanol, secondary butyl alcohol, isoamylalcohol and tetrahydrofuran.

The double salts of SAM obtained according to the present invention canbe preserved indefinitely in the dry state, as stated, practicallyunaltered.

The following examples illustrate the method of preparation of the newsalts according to the invention, it being however understood that theseexamples are purely illustrative and do not limit the invention.

The process discovered by us does not require temperatures higher thanambient temperature in any stage, and is carried out in an acidenvironment. This is very important in avoiding the decomposition of theSAM. Furthermore it does not require complicated and laborious processessuch as chromatography over columns of ion exchange resin or carbon,which would make it difficult to carry out industrially.

EXAMPLE 1

To 90 kg of yeast enriched in SAM (6.88 g/kg) in accordance with Schlenk(Enzymologia 29, 283 (1965)) are added 11 l of ethyl acetate and 11 l ofwater at ambient temperature. After energetic agitation for 30 minutes,50 l of 0.35 N sulphuric acid are added, continuing agitation for afurther hour and a half. After filtering and washing with water, 140 lof solution are obtained containing 4.40 g/l of SAM, equal to 99.5% ofthat present in the starting material.

A solution of 2.3 kg of picrolonic acid in 24 liters ofmethylethylketone is added to the previous solution under agitation.After standing for one night, the precipitate is separated bycentrifuging and washed with water.

The solid thus obtained is added under agitation to a mixture of 18liters of a 0.1 N solution of sulphuric acid and p-toluenesulphonicacid, and 18 liters of methylethylketone. After standing, the organicphase separates and is fed to the picrolonic acid recovery system, theaqueous phase is treated with a little methylethylketone to eliminateresidual traces of picrolonic acid, decolouring charcoal is added and itis then filtered.

This solution (16.5 l) contains 33.8 g/l of SAM, equal to 90% of thecompound present in the yeast. When analysed by thin layerchromatography in accordance with Anal. Biochem. 4, 16-28 (1971) it isknown to contain only SAM without traces of its decomposition productsor other organic bases.

The above solution is poured into 100 liters of acetone under agitation.

After standing, it is decanted from the solvent and the solid isdissolved in 3.3 kg of a 15% solution of p-toluenesulphonic acid inmethanol. After adding decolouring charcoal, the mixture is filtered andadded to 25 l of ethyl ether.

1184 g of a well crystalline salt precipitate which is easilyfilterable, not very hygroscopic, and very soluble in water (more than20%) with the formation of a colourless solution. The salt is onlyslightly soluble in methanol and ethanol, and insoluble in acetone,methylethylketone, chloroform, higher alcohols and benzene. From thinlayer chromatography in accordance with Anal. Biochem. 4, 16-28 (1971)the product is shown to be free from any impurity.

Centesimal analysis of the salt gave the following results: C = 36.39%,H = 4.6%, S = 16.7%, N = 8.8%; The calculated values for C₂₉ H₄₂ N₆ R₁₉S₅ (M.W. 938.98) are: C = 37.09%, H = 4.51%, S = 17.7%, N =8.95%.Moreover: H₂ SO₄ = 20.5% P-toluensulphonic acid = 36.0% SAM =41.7%Calculated: H₂ SO₄ = 20.89% P-toluensulphonic acid = 36.67% SAM =41.54%

Humidity determined in accordance with K. Fischer: 1.7-2%

The U.V. spectrum of the new compound shows an absorption maximum at 260nm, E₁ ^(1%) _(cm) = 182.

This data agrees with a compound of formula ##EQU2##

The new compound has further been identified by the enzymatic methodbased on the enzymatic methylation of nicotine amide or guanidine aceticacid with SAM (G. L. Cantoni, J. Biol. Chem., 189, 745 (1951); G. De LaHoba, B. A. Jamieson, S. H. Mudd and H. H. Richards, J. Amer. Chem. Soc.81, 3975 (1959).

EXAMPLE 2

1.15 kg of picrolonic acid dissolved in 10 liters of isobutyl alcoholare added to 70 liters of solution originating from the lysis of yeastcells, obtained by using the same raw material and same method asexample 1.

After standing for one night, the precipitate formed is separated bycentrifuging.

The separated solid is treated with 9 liters of a 0.1 N solution ofsulphuric acid and p-toluenesulphonic acid, and with 9 liters ofmethylisobutylketone.

After standing, the organic phase is separated, while the aqueous phaseis freed from traces of picrolonic acid by washing with a littlemethylisobutylketone. Decolouring charcoal is added and the mixture isfiltered, pouring the filtrate into 70 liters of methyl alcohol. Afterstanding, the solid formed is decanted from the solvent and is dissolvedin a 15% solution of p-toluenesulphonic acid in methanol (1.65 kg).

After decolouring with charcoal, the filtrate is added to 10 liters ofchloroform to obtain an easily filterable crystalline precipitate of SAMdisulphase di-p-toluenesulphonate.

The product obtained (1110 g) gave on analysis results which wereidentical to the product of example 1.

EXAMPLE 3

1.15 kg of picrolonic acid dissolved in 12 liters of n-butanol are addedto 70 liters of solution originating from the lysis of yeast cells,obtained by the same lysis method and with the same raw material asexample 1. After standing for 1 night, the precipitate is separated bycentrifuging.

The solid obtained is divided between 9 liters of a 0.1 N solution ofsulphuric acid and p-toluenesulphonic acid, and 12 liters of n-butanol.

After standing, the organic phase separates, while the aqueous phase isfreed traces of picrolonic acid by washing with a little n-butanol.

Decolouring charcoal is then added and the mixture is filtered, pouringthe filtrate into 65 liters of n-propyl alcohol. After standing, theprecipitated solid is decanted from the solvent and dissolved in a 15%solution of p-toluenesulphonic acid in methanol (1.65 kg).

After decolouring with charcoal, the filtrate is poured into 14 litersof n-butanol to obtain a well filterable crystalline precipitate of SAMdisulphate di-p-toluenesulphonate.

The final product (1155 g) gave the same results on analysis as theproduct of example 1.

EXAMPLE 4 Purification of the specific enzyme

50 ml of Sepharose (polysaccharide produced by Pharmacia Fine ChemicalsAB, Uppsala -- Sweden) caked and suspended in water, are treated withcyanogen bromide in accordance with the known methods for bondingsubstances containing amine groups to matrices consisting ofpolysaccharide gel.

An excess of L-lysine is added to the gel so prepared. It is repeatedlywashed after the reaction with distilled water, with a pH 8.5 buffermixture and with a pH 4.5 buffer mixture. The gel is then used forpacking a column having a diameter of 1.5 cm and a height of 30 cm. Abuffer mixture of 0.05 M triethanolamine and 0.01 M H₂ SO₄ of pH 8.0 ispassed through the column until complete equilibration is obtained.

2 ml of yeast extract containing the specific enzyme are laid on thecolumn, obtained by sonification or by homogenising with dry ice andpossibly after enrichment with the specific enzyme. The column is theneluted with the same buffer mixture as used for equilibration, followingthe distribution of the proteins in the eluate by ultravioletspectrophotometric measurements. Simultaneously the synthetasis activityis measured in the various fractions in accordance with J. A. Stekol,Methods in Enzymology, vol. 6, pag. 566 (1963). Those fractionsdisplaying a relevant synthesis activity are added together and thesolution so obtained shows a specific activity at least 20 times greaterthan the raw extract. The enzyme can be further concentrated in thissolution by precipitation with salts, with organic solvents or inaccordance with other known methods for concentrating protein solutions.

Preparation of SAM

30 ml of caked Sepharose gel are activated with cyanogen bromide or byany other known method for bonding proteins to polysaccharide gelmatrices.

4 ml of a solution of the specific enzyme purified as above andcontaining about 100 mg of protein are added to the activated gel. Thesuspension of activated gel and the enzyme solution are agitated at 4°Cfor 18 hours.

The resin is washed with water. This wash liquid contains about 70% ofthe total enzymatic activity initially present in the solution of thespecific enzyme. By incubating the Sepharose prepared as above with theaforementioned method for determining synthetasis activity, it isobserved that approximately 20% of the total activity is bonded to thepolysaccharide.

The Sepharose prepared as above is used for packing a column having adiameter of 1.5 cm and a height of 20 cm. A solution containing 0.675 Mtriethanolamine, 0.150 M magnesium sulphate, 0.05 M ATP, 0.05 MM-methionine, and 0.01 M KCl is passed through the column at a speed of5 ml per hour and at a temperature of 25°-27°.

The eluate from the column analysed for SAM content shows that theconversion yield is 30%.

Preparation of double salts of SAM with sulphuric acid andp-toluenesulphonic acid

103 ml of eluate containing 6 g/l of SAM are acidified with H₂ SO₄ untila pH of 3 is reached, and to this is added under agitation a solution of2.3 g of picrolonic acid in 25 ml of methylethylketone. After standingfor one night, the precipitate is filtered and washed with water. Theprecipitate is redissolved in 18 ml of a 0.1 N solution of H₂ SO₄ andp-toluenesulphonic acid, and in 18 ml of methylethylketone.

After agitation and standing, the organic layer is separated and theaqueous layer is treated with a little methylethylketone to eliminatethe last traces of picrolonic acid. After separating the water layer,decolouring charcoal is added and the mixture is filtered. 16.5 ml ofaqueous colourless solution are obtained, which contains 33.8 g/l of SAMequal therefore to 90% of the SAM contained in the original solution. Onthin layer chromatography analysis, the solution is shown to containonly SAM. 16.5 ml of the solution are poured into 100 ml of acetone.After agitation and standing, the liquid is separated by decanting. Thesolid is dissolved in 6.6 g of a 15% solution of p-toluenesulphonic acidin methanol. After adding decolouring charcoal and filtering, thesolution is poured into 25 ml of ethyl ether. It is filtered afterstanding, and the well crystalline salt obtained weights 0.967 g and hasthe composition:

SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃ C₆ H₄ SO₃ H. On analysis: calculated for C₂₂ H₃₄O₁₆ N₆ S₄ : C: 34.46%, H: 4.47%, N: 10.96%, S: 16.72%; found: C: 33.68%,H: 4.65%, N: 10.8%, S: 16.5%; SAM^(+:) 50.5%, H₂ O: 2.1%, H₂ SO₄ :25.3%, p-toluenesulphonic acid: 21.7%.

The ultraviolet spectrum shows a maximum at 260 nm, E₁ ^(1%) _(cm) =179. On repeating the process in an identical manner, but using 3.3 g ofa 15% solution of p-toluenesulphonic acid in methanol, in the subsequentprecipitation stage using 25 ml of ethyl ether, 1.18 g of the saltSAM⁺.H₂ SO₄.2CH₃ C₆ H₄ SO₃ H is obtained having identicalcharacteristics with those indicated for the product of example 1.

For some years it has been known from biochemical research that SAM isthe only specific donot of methyls in living organisms for thebiochemical reactions of transfer of the CH₃ group, which arefundamental reactions in the lipidic, protidic and glucidic metabolism.

By way of example we give below some of the most important SAM-dependenttransmethylation reactions:

a. N-transmethylation: adenine, carnitine, carnosine, creatine,2,6-diaminopurine, adrenaline, guanine, hordenine, N'-nicotinamide,phosphatidilcoline, ricinine;

b. O-transmethylation: N-acetylserotonine, dopamine, epinine,d-adrenaline, 1-adrenaline, ergosterol, 1-noradrenaline, pectine,ubiquinone;

c. S-transmethylation: 2,3-dimercaptopropanol, H₂ S, methionine,methylmercaptan, S-mercaptopropionic acid, S-mercaptoethanol,thiopyrimidine, thiouracyl;

d. C-transmethylation: cytosine, thymine.

This means, referring in particular to the human organism, that SAM actsin the following metabolic processes:

Biosynthesis of choline; biosynthesis of phosphatidylcoline; activity ofenzymes which require SH groups; metabolism of catecholamines;metabolism of biogene centroencephalic amines; metabolism of serotonine;metabolism of histamine; metabolism of vitamin B12 and of pholic acid;metabolism of creatine; metabolism of myosine; metabolism of histones;metabolism of RNA; metabolism of DNA; metabolism of protein substances;metabolism of some hormones of cyclopentane perhydrophenantrenicnucleus, the main ones of which are the estrogens; metabolism of thetriglycerides.

It has also been known for some time that SAM, once demethylated by themethyltransferasic enzymes, is transformed into S-adenosilhomocysteine(SAO) which is an indirect donor of hydrosulphide groups and hence has adetermining importance in the metabolism of all compounds which requireSH groups for carrying out their biological activity.

Particularly important among these are some thioenzymes and thesulphurated amino acids.

SAO in its turn is decarboxylated in the organism, and thedecarboxylated product is the principal donor of the aminopropyl group,indispensible -- according to the most recent biochemical knowledge --for the biosynthesis of polyamines. The process is catalysed by variousenzymes among which a specific one is aminopropyl-transferasis.

Summarizing we may say that it is known that SAM in the human organismis closely connected with all biochemical reactions of:

A. transmethylation (specific yielding of the CH₃ group)

B. transsulphuration (specific yielding of the SH group)

C. transaminopropylation (specific yielding of the aminopropyl group).

The sum of this knowledge could lead one to think that SAM could havesome therapeutic action in the treatment of pathological states linkedwith the shortage or other deficiency conditions in the organism withrespect to some of the many products mentioned above.

However the extreme instability of SAM and the lack, up to the presenttime, of any method for making it stable for sufficient times undernormal ambient conditions has prevented this product from being givenany pharmacological or clinical tests and hence has prevented anypractical use being found for it in the field of human therapy.

Only after the preparation of the new SAM salts according to the presentinvention, salts which are practically indefinitely stable at ambienttemperature, has it been possible to carry out a systematicpharmacological and clinical study which has led to the discovery forthe new salts of therapeutic properties completely surprising in theirquality and intensity.

From the enormous quantity of pharmacological and clinical datacollected for this new product, we give hereinafter only some elementssufficient to clearly indicate to experts in the art the essentialcharacteristics of the new product and its main uses in human therapy.

For simplicity hereinafter we shall indicate simply by "SAM salt" thetwo double salts according to the invention, because of their absoluteidentical use.

In the pharmacological and clinical data we indicate for the sake ofsimplicity with the abbreviation SAM the salt

Sam⁺.hso₄ ⁻.h₂ so₄ .2ch₃ c₆ h₄ so₃ h.

also the administered amounts are always of

Sam⁺.hso₄ ⁻.h₂ so₄ .2ch₃ c₆ h₄ so₃ h.

however the data may refer equally well to the other double salt.

TOXICITY -- the SAM salt according to the invention has provedabsolutely free from acute toxicity, chronic toxicity, local intoleranceor secondary effects.

In particular, the DL₅₀ in the mouse is greater than 6 g/kg/os and 2.5g/kg/i.p.

The tolerability and chronic toxicity tests were carried out on rats ofthe Wistar and Sprague-Dowley stock administering for 12 months 10-20mg/kg per day of product: at the end of the treatment the various organsand apparatus showed no pathological alteration.

The tetratogenesis tests were carried out on rabbits and rats: even withthe administration of massive doses of SAM, approximately 10 times themaximum therapeutical doses, no tetratogenic actions were encountered orany malformations in the embryons or terminal feti.

The addition of doses up to 0.1-0.2 mg/ml of product in survivingcultures of human lymphocytes or hepatic mouse cells does not productany change in the blasticising index for the cellular elements.

The intravenous administration of doses up to 40 mg/kg does not produceany pyrogenic manifestation in the rabbit.

The venous administration in the rabbit and cat of 40 mg/kg doses doesnot cause any change in the carotid pressure, the cardiac andrespiratory frequency or the electrocardiac trace.

The local tolerability of the intermuscular injection, even afteradministrations repeated over 180 days, and of the intravenous injectionin the marginal vein of the auricular pavillon of the rabbit, isexcellent.

In man, in young volunteer healthy subjectes of both sexes subjected toadministration by the rapid intravenous method or by phleboclysis ofdoses of SAM equal to 10-300 mg/ (average weight 70 kg), thesimultaneous examination of the minimum and maximum pressure and of thepulse and respiratory frequency at 1,5,15,20,30,60 minutes and at2,3,6,8,10,12,24 hours from the end of administration does not show anyvariation from normal values.

The electrocardiograph trace does not show any variations in the PQinterval, in the ST section, nor any appearance of extrasistol or otheralterations at 30", 1', 2', 3', 5', 10' and 20' from administration.

In the hemopoietic apparatus and in the hepatic and renal operationthere were no variations which were statistically significant fromnormal.

PHARMACOLOGY

We would repeat here that each time we speak hereinafter of theadministration of SAM, we mean that the following has been administered:

Sam⁺.hso₄ ⁻.h₂ so₄ .2 ch₃ c₆ h₄ so₃ h and/or SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃ C₆H₄ SO₃ H. In order to indicatively determine how SAM is distributed inthe tissues, S-Adenosilmethionine (Methyl C¹⁴) was prepared. Thedistribution of this product in rats was studied by administering a doseof 10 mg/kg/e.v. equal to 24 μci of radioactive product. The specificactivity of the product was 58 mCi/m moles. Parallel to this, anautoradiographic study was made on the mouse. The results of these twoexperiences show that SAM is distributed very rapidly to all thetissues.

We give by wey of example a part of the data relative to each of theorgans considered:

Distribution of SAM in some rat tissues.

The values are expressed as μgr/gr.

    ______________________________________                                        Tissue   15'      1 h      4 h    8 h    24 h                                 ______________________________________                                        Liver    4.02     7.47     13.1   13.4   13.5                                 Suprarenal                                                                             4.26     8.46     11.7   10.8   10.9                                 glands                                                                        Spleen   3.15     2.96     8.1    6.2    6.7                                  Hypophysis                                                                             5.6      5.8      19.5   11.3   10.3                                 Hypothalamus                                                                           0.7      1.5      2.4    3.0    3.2                                  Rind     0.6      1.1      1.8    2.1    2.3                                  Plasma   18.6     5.2      5.3    4.5    3.1                                  ______________________________________                                    

It was consequently deduced that the new salts according to the presentinvention donate the CH₃ group to all the tissues provided withmethyltransferasis activity. In other words the capacity of the newproducts according to the invention to electively localise themselves inall the organs provided with methyltransferasis systems was deduced.

This was confirmed by successive pharmacological tests. A whole seriesof tests carried out on rats has shown that the new compounds exercise aconsiderable protective and resolutive action in hepatic steatosis byhyperlipidic-hyperproteic diet according to Handler, in steatosis byacute alcoholic intoxication and by other toxic agents (carbontetrachloride, bromobenzene etc.) by the administration of only 15mg/kg/i.p.; both from the morphohistochemical and analytical point ofview, the SAM significantly reduced the accumulation of lipids at thehepatocite level while it favours the restoration of normal levels ofphospholipids reduced after intoxication with CCl₄.

Hepatic phospholipids in rats after intoxication with CCl₄ and treatmentwith SAM.

    ______________________________________                                        Treatment          Total phospholipids (mg/g)                                 ______________________________________                                        Physiological solution                                                                           30.57 ± 1.18                                            CCl.sub.4          18.87 ± 1.06                                            CCl.sub.4 + SAM 15 mg/kg/i.p.                                                                    27.20 ± 1.25                                            CCl.sub.4 + SAM 150 mg/kg/i.p.                                                                   20.87 ± 0.42                                            CCl.sub.4 + Ad-Met 15 mg/kg/i.p.                                                                 19.9   ± 0.92                                           ______________________________________                                    

The values are the average ± E.S. of 10 values for each group.

In studying the hepatoprotective activity we have used an experimentaldevice which produces in the rat the so-called hepatic cholesteroldegeneration (Ridout and Coll., Biochem. J. 52, 99, 1952).

In this method, by means of a suitable diet, a conspicuous increase inthe total hepatic fats and hepatic cholesterol are obtained in theanimals. The substances which act in the lipidic metabolism reduce orannul this increase.

The animals were divided into six groups. The first group wasadministered with a diet which was varied at will. The second group wasadministered with the basic diet of Ridout (20 g per rat per day); theother groups were administered with the same diet in the same doses butenriched with cholesterol to the extent of 0.2 g/rat/day. The treatmentlasted 3 weeks.

The groups 4,5,6 were administered with SAM in the following doses:1,2,5 mg/kg/i.p. per day.

At the end of 3 weeks all the animals were killed, the livers werewithdrawn and the total fats (Best and Coll., Biochem. J. 40, 368, 1966)and cholesterol (Sperry and Brand, J. Biol. Chem. 150, 315, 1943) weredetermined.

The results showed that the batches subjected to treatment with SAM indoses of 1-2 mg/kg/i.p. were poorly protected, while the batch treatedwith 5 mg/kg/i.e. was completely protected.

Hepatic cholesterol and total fats at the end of the experiment (averageper batch)

    Batch   Fresh liver                                                                              Total fats    Cholesterol                                  weight g       g       %         mg    %                                      ______________________________________                                        I       15         1.41     9.4    40    2.6                                  II      18         1.93    10.6    68    3.7                                  III     16         3.84    24.0    92    5.7                                  IV      17         3.70    21.6    90    5.2                                  V       16         3.5     21.9    67    4.1                                  VI      16         2.0     12.5    61    3.8                                  ______________________________________                                    

Another pharmacological aspect investigated by us was theantiinflammatory and analgesic effects of SAM.

Ot the various tests we shall mention the most classic, namely the edemaby carragenine and by egg white as a test for acute inflammation; andgranuloma by cotton peelets and arthritis by adjuvant as a test forchronic inflammation. In all cases SAM proved active both administeredorally (dose between 20 and 100 mg/kg) and parenterally (doses between10 and 20 mg/kg) in comparison with other known pharmaceutical products(Ibuprofen-Indometacina). The analgesia tests were in the form of thehot plate tests and stretching by acetic acid, and the Randal andDrlitto tests in the rat. The pharmaceutical product also proved activein these tests in comparison with known pharmaceutical products studied.

A further aspect considered by us was the possible action of SAM on thesleeping time by barbiturates.

For this purpose an experiment was made in which groups of mice receivedhexobarbital in a dose of 80 mg/kg/i.p. in accordance with the method ofHolten and Larsen (Acta Pharmacol. Toxicol. 1956, 12, 346); one groupwas the control group and the second received SAM in a dose of 10mg/kg/i.p. (table).

    ______________________________________                                                         Sleeping time (min.)                                         Controls         24.4 ± 2.7                                                SAM 10 mg/kg/i.p.                                                                              41.2 ± 5.8                                                ______________________________________                                    

An examination of the data showed that SAM is active in prolonging thesleeping time induced by hexobarbital.

CLINICAL TESTS

Where the administration of SAM is mentioned hereinafter, this signifiesthe administration of SAM⁺.HSO₄ ⁻.H₂ SO₄ .2CH₃ C₆ H₄ SO₃ H and/orSAM⁺.HSO₄ ⁻ .H₂ SO₄ CH₃ C₆ H₄ SO₃ H.

Following the information gained from the pharmacological tests, theclinical tests were orientated on morbid affections in which thefollowing appear primitively or secondarily altered:

1 -- the metabolism of lipids

2 -- the metabolism of protids and glucids

3 -- the metabolism of catecholamines and the biogene amines.

1. From tests conducted clinically on hundreds of subjects using dosesof SAM varying over a very wide interval, it was found that the newcompound induces a rapid fall in the hepatic lipids in thehepatosteatosis of the most varied pathogenesis, identifiable by abioptic examination repeated after the end of the treatment cycle, evenafter 60 days from the end of treatment. The administration of theproduct also induces a marked fall in the high values of totalcholesterolemia, of hypertriglyceridemia and normalises the altered β/αlipoproteic ratios in subjects with hyperdislipidemia in theuncompensated stage.

This hypocholesterolemising and hypolipemising action is verified evenin doses of about 20-30 mg administered 2-3 times per day, and isproportional to the dose.

In clear arteriosclerosis with clinical manifestations of thepsychoaffective sphere, with turbemnesics and secondarycentroencephalics (determination by arteriosclerotic encephalopathia)and phenomena of cerabral hypoxia, the administration of SAM byintramuscular or, in graver cases, by intravenous injection or by lowphleboclysis, in doses between 20 and 40 mg 3-4 times per day, has showna very favourable modification of the sumptomatology.

In particular, in clear hypoxydotic states the recovery of the functionsrelated to the like of relationship was very quick and statisticallysignificant.

In post-apoplectic syndromes a greater rapidity was found in theimprovement of the clinical framework.

2. Hundreds of subjects were treated clinically affected with: secondaryhypoprotidemias and disprotidemias; persistent and aggressive cronichepatopathias; precyrrotic and cyrrotic states; malabsorption syndromes,protide dispersing syndromes. The administration of doses variablebetween 20 and 200 mg of SAM per day by intermuscular or intravenousinjection or orally, according to the gravity of the case, caused astatistically significant increase in the total protidemia, an increasein the albumin amount and a tendency to normalise the altered percentageratios between the electrophoretic fractions of the serum. This proteinanabolising activity was followed by an often very important improvementin the subjective symptomology and the general objective conditions, andby the normalizing of all the tests of hepatic functionality.

3. Particularly surprising results were obtained in clinicalapplications of the new enzymatic salt according to the invention, inwhich morbid frameworks existed which were clearly correlated withmodifications in the exchange of biogene amines, for example:

a. pathological frameworks of neuropsychiatric pertinence;

b. Parkinsons disease and Parkinsonism of various eziopathogeneses;

c. Antiphologistic and analgesic action in the treatment of osteoarthritis, and antalgic activity in certain neurological manifestations;

d. Disturbances of the sleeping-waking rhythm.

With regard to point (a), a vast clinical casuistry conducted byexamining the clinical behaviour and the tests of Hamilton andWittenberg, has clearly shown that the administration of doses varyingbetween 20 and 50 mg of SAM 3-4 times per day for a period of 5-15 daysinduces, excluding any other form of therapy, a significant remission ofthe main parameters considered for the diagnosis of depressive forms.

With regard to point (b) relative to the treatment for Parkinsonsdisease and Parkinsonisms, it has been found that:

The administration of SAM in doses of 10-40 mg per day by intramuscularor intravenous injection, or orally -- according to the gravity of thecase -- in association with the habitual therapy with Levodopa, givesrise to a statistically more significant improvement in the akinesia andrigidity with respect to that which occurs in patients treated only withLevodopa. favourable modifications are also found in the extent of theParkinson tremor, which cannot be modified by Levodopa alone.

The administration of SAM distinctly improves the Levodopa-dependentpsychic disturbances, with particular regard to depressive states andpsychic manifestations of irritative type.

The administration of SAM in the aforementioned doses significantlyblocks the train of Levodopa side effects of the various organs andapparatus, with particular regard to neusea, vomit, inappetite,hypotension, asthenia, cephalea, hypersudoration and insomina.

With regard to point (c), SAM, which pharmacological results show tohave intense antiphlogistic and analgesic activity, has proved active inall osteoarthritic forms treated with a dose of 30 mg twice a day byintermuscular or intravenous injection, and 30-50 mg orally 4 times perday.

After only 7 days of treatment, the muscular spasm, the limitation ofmovement, localised pain, and rigidity were influenced in astatistically significant manner, with respect to the placebo. No caseof gastric pyrosis was observed in 90 cases treated. The search forconcealed blood in the feces never showed any modification during thetreatment.

SAM, compared with a non-steroid antiphlogistic drug commonly used in adouble blind study, proved to possess a therapeutic efficiency equal toindomethacin.

The antalgic activity of SAM was also tested in different subjects withdifferent neurological frameworks: neuritis, polyneuritis, anthralgia,sciatica, radiocolitis, torticollis. The therapeutic effect wasavailable and efficient from the first day of administration of a doseof 15 mg twice a day by intermuscular injection or 30-50 mg 3-4 timesper day orally. Analogous results were obtained in subjects withrecurring and resistant cephalalgia with the administration of the drugorally in masticable tablets.

With regard to point (c), i.e. disturbances of the sleeping-wakingrhythm, with particular regard to insomnia, the new product according tothe invention is able with a dose of 10-30 mg orally, to considerablyimprove the altered sleeping-waking ratios by inducing a physiologicalsleep without recurrence to the use of barbiturates or other substancesof cortical and centroencephalic depressive action.

From that summarized heretofore the numerous unexpected perspectivesopened up by the new drug in the field of human therapy are evident.

Summarizing, we can say that the fields of use already ascertained are:

treatment of hepatopias, hyperdislipidemias, generalised or localarteriosclerosis, psychiatric manifestations of depressive andneurological type, degenerative arthromathies, neurological algicmanifestations and disturbances of the sleeping-waking rhythm, whereasmany other fields of use still remain to be examined and ascertained.

The new SAM salts are preferably administered by intramuscular orintravenous injection, or in oral or sublingual tablets, or in capsules.

Some pharmaceutical compositions are given below:

    a)  A 400 mg tablet contains                                                      SAM salt               66.66       mg                                         Excipients:                                                                   Starch                                                                        Lactose                                                                       Magnesium stearate                                                            Talc                                                                          Aroma                  q.n. 400    mg                                     b)  A 250 mg capsule contains                                                     SAM salt               66.66       mg                                         Excipients:                                                                   Starch                                                                        Lactose                                                                       Magnesium stearate                                                            Na.sub.3 PO.sub.4      q.n.250     mg                                     c)  A lyophilised phial contains                                                  SAM salt               11.11       mg                                         A muscular solvent phial contains                                             Lidocaine              20          mg                                         Solution of phosphate buffers                                                                        q.n. to 3   ml                                     d)  A lyophilised phial contains                                                  SAM salt               33.33       mg                                         A muscular solvent phial contains                                             Lidocaine              20          mg                                         Solution of phosphate buffers                                                                        q.n. to 3   ml                                     e)  A 2 g suppository contains                                                    SAM salt               111.11      mg                                         Suppository mass       q.n. to 2.0 g                                  

Other forms of administration may be:

a. Drinkable bottles

b. Liquids for ocular instillation

c. Liquids for intranasal instillation

d. Liquids for aerosol or spray application

e. Liquids and ointments for topical use in which the active principleis diluted in the normal acceptable pharmaceutical solvents ("TecnologiaFarmaceutica" -- Second Volume, Silvano Casadia -- Second Edition -- ed.Cisalpino-Goliardica -- Milan -- 1972).

In conclusion we may say that the therapeutic doses of SAM lie between 5and 300 mg per day, according to the particular type and gravity of theaffection treated.

Larger doses may be used if necessary in view of the absolute absence oftoxicity of the salts according to the invention.

What is claimed is:
 1. Double salts of S-adenosil-L-methionine (SAM)with sulphuric acid and p-toluenesulphonic acid.
 2. Double salt asclaimed in claim 1 corresponding to the formula SAM⁺.HSO₄ ⁻.H₂ SO₄ .2CH₃ C₆ H₄ SO₃ H.
 3. Double salt as claimed in claim 1 corresponding tothe formula SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃ C₆ H₄ SO₃ H.
 4. Process for preparingdouble salts of S-adenosil-L-methionine (SAM) with sulphuric acid andp-toluensulphonic acid in whicha. a concentrated solution of SAM isprepared; b. the SAM present in the solution is precipitated byacidifying with a solution of picrolonic acid; c. the SAM picrolonate isdissolved in a mixture consisting of equal parts by volume of an aqueoussolution of equal normalities of p-toluenesulphonic acid and sulphuricacid, and an organic solvent partially miscible with water; d. the SAMsalt is precipitated from the aqueous layer by a ketone or alcohol basedsolvent totally soluble in water; e. the precipitated salt is dissolvedin a solution of p-toluenesulphonic acid in methanol; f. the double saltof SAM with sulphuric acid and p-toluenesulphonic acid is precipitatedby a suitable organic solvent.
 5. Process as claimed in claim 4, inwhich the stage (a) is carried out by the lysis of yeast cells bytreatment with ethyl acetate and sulphuric acid at ambient temperature.6. Process as claimed in claim 4, in which the stage b. is carried outusing solutions of picrolonic acid in water or in an organic solventsoluble in water.
 7. Process as claimed in claim 4, in which the stagec. is carried out using aqueous solutions of equal normalities ofsulphuric acid and p-toluenesulphonic acid, both lying between 0.05 and0.2.
 8. Process as claimed in claim 4, in which the stage d. is carriedout using 4-8 volumes, with respect to the aqueous solution, of anorganic solvent chosen from the group consisting of acetone, methylalcohol, ethyl alchol, propyl alcohol.
 9. Process as claimed in claim 4,in which the stage e. is carried out using 10-4% solutions ofp-tolurnsulphonic acid in methanol.
 10. Process as claimed in claim 9,in which the minimum quantity of methanol necessary for dissolving thesalt is used.
 11. Process as claimed in claim 9 wherein the methanol isused in a volume equal to at least double that necessary to dissolve theprecipitate originating from stage d.
 12. Process as claimed in claim10, in which the double salt SAM⁺.HSO₄ ⁻.H₂ SO₄ .2CH₃ C₆ H₄ SO₃ Hprecipitates during treatment with the organic solvent in accordancewith stage f.
 13. Process as claimed in claim 11, in which the doublesalt SAM⁺.HSO₄ ⁻.H₂ SO₄.CH₃ C₆ H₄ SO₃ H precipitates during treatmentwith the organic solvent in accordance with stage f.